Deletion of CD39 on natural killer cells attenuates hepatic ischemia/reperfusion injury in mice

Natural killer (NK) cells play crucial roles in innate immunity and express CD39 (Ecto-nucleoside triphosphate diphosphohydrolase 1 [E-NTPD1]), a rate-limiting ectonucleotidase in the phosphohydrolysis of extracellular nucleotides to adenosine. We have studied the effects of CD39 gene deletion on NK cells in dictating outcomes after partial hepatic ischemia/reperfusion injury (IRI). We show in mice that gene deletion of CD39 is associated with marked decreases in phosphohydrolysis of adenosine triphosphate (ATP) and adenosine diphosphate to adenosine monophosphate on NK cells, thereby modulating the type-2 purinergic (P2) receptors demonstrated on these cells. We note that CD39-null mice are protected from acute vascular injury after single-lobe warm IRI, and, relative to control wild-type mice, display significantly less elevation of aminotransferases with less pronounced histopathological changes associated with IRI. Selective adoptive transfers of immune cells into Rag2/common gamma null mice (deficient in T cells, B cells, and NK/NKT cells) suggest that it is CD39 deletion on NK cells that provides end-organ protection, which is comparable to that seen in the absence of interferon gamma. Indeed, NK effector mechanisms such as interferon gamma secretion are inhibited by P2 receptor activation in vitro. Specifically, ATPgammaS (a nonhydrolyzable ATP analog) inhibits secretion of interferon gamma by NK cells in response to interleukin-12 and interleukin-18, providing a mechanistic link between CD39 deletion and altered cytokine secretion. CONCLUSION: We propose that CD39 deficiency and changes in P2 receptor activation abrogate secretion of interferon gamma by NK cells in response to inflammatory mediators, thereby limiting tissue damage mediated by these innate immune cells during IRI.

Mixture of periodontopathogenic bacteria influences interaction with KB cells

INTRODUCTION: The purpose of this study was to investigate the adhesion and invasion of periodontopathogenic bacteria in varied mixed infections and the release of interleukins from an epithelial cell line (KB cells). METHODS: KB cells were co-cultured with Porphyromonas gingivalis ATCC 33277 and M5-1-2, Tannerella forsythia ATCC 43037, Treponema denticola ATCC 35405 and Fusobacterium nucleatum ATCC 25586 in single and mixed infections. The numbers of adherent and internalized bacteria were determined up to 18 h after bacterial exposure. Additionally, the mRNA expression and concentrations of released interleukin (IL)-6 and IL-8 were measured. RESULTS: All periodontopathogenic bacteria adhered and internalized in different numbers to KB cells, but individually without any evidence of co-aggregation also to F. nucleatum. High levels of epithelial mRNA of IL-6 and IL-8 were detectable after all bacterial challenges. After the mixed infection of P. gingivalis ATCC 33277 and F. nucleatum ATCC 25586 the highest levels of released interleukins were found. No IL-6 and IL-8 were detectable after the mixed infection of P. gingivalis M5-1-2 and F. nucleatum ATCC 25586 and the fourfold infection of P. gingivalis ATCC 33277, T. denticola ATCC 35405, T. forsythia ATCC 43037 and F. nucleatum ATCC 25586. CONCLUSION: Anaerobic periodontopathogenic bacteria promote the release of IL-6 and IL-8 by epithelial cells. Despite a continuous epithelial expression of IL-8 mRNA by all bacterial infections these effects are temporary because of the time-dependent degradation of cytokines by bacterial proteases. Mixed infections have a stronger virulence potential than single bacteria. Further research is necessary to evaluate the role of mixed infections and biofilms in the pathogenesis of periodontitis.

Anti insulin-like growth factor I receptor immunoliposomes: a single formulation combining two anticancer treatments with enhanced therapeutic efficiency

Context: Through overexpression and aberrant activation in many human tumors, the IGF system plays a key role in tumor development and tumor cell proliferation. Different strategies targeting IGF-I receptor (IGFI-R) have been developed, and recent studies demonstrated that combined treatments with cytostatic drugs enhance the potency of anti-IGFI-R therapies. Objective: The objective of the study was to examine the IGFI-R expression status in neuroendocrine tumors of the gastroenteropancreatic system (GEP-NETs) in comparison with healthy tissues and use potential overexpression as a target for novel anti-IGFI-R immunoliposomes. Experimental Design: A human tumor tissue array and samples from different normal tissues were investigated by immunohistochemistry. An IGFI-R antagonistic antibody (1H7) was coupled to the surface of sterically stabilized liposomes loaded with doxorubicin. Cell lines from different tumor entities were investigated for liposomal association studies in vitro. For in vivo experiments, neuroendocrine tumor xenografts were used for evaluation of pharmacokinetic and therapeutic properties of the novel compound. Results: Immunohistochemistry revealed significant IGFI-R overexpression in all investigated GEP-NETs (n = 59; staining index, 229.1 +/- 3.1%) in comparison with normal tissues (115.7 +/- 3.7%). Furthermore, anti-IGFI-R immunoliposomes displayed specific tumor cell association (44.2 +/- 1.6% vs. IgG liposomes, 0.8 +/- 0.3%; P < 0.0001) and internalization in human neuroendocrine tumor cells in vitro and superior antitumor efficacy in vivo (life span 31.5 +/- 2.2 d vs. untreated control, 19 +/- 0.6, P = 0.008). Conclusion: IGFI-R overexpression seems to be a common characteristic of otherwise heterogenous NETs. Novel anti-IGFI-R immunoliposomes have been developed and successfully tested in a preclinical model for human GEP-NETs. Moreover in vitro experiments indicate that usage of this agent could also present a promising approach for other tumor entities.

Glucocorticoids protect renal mesangial cells from apoptosis by increasing cellular sphingosine-1-phosphate

Neutral ceramidase (NCDase) and sphingosine kinases (SphKs) are key enzymes regulating cellular sphingosine-1-phosphate (S1P) levels. In this study we found that stress factor-induced apoptosis of rat renal mesangial cells was significantly reduced by dexamethasone treatment. Concomitantly, dexamethasone increased cellular S1P levels, suggesting an activation of sphingolipid-metabolizing enzymes. The cell-protective effect of glucocorticoids was reversed by a SphK inhibitor, was completely absent in SphK1-deficient cells, and was associated with upregulated mRNA and protein expression of NCDase and SphK1. Additionally, in vivo experiments in mice showed that dexamethasone also upregulated SphK1 mRNA and activity, and NCDase protein expression in the kidney. Fragments (2285, 1724, and 1126 bp) of the rat NCDase promoter linked to a luciferase reporter were transfected into rat kidney fibroblasts and mesangial cells. There was enhanced NCDase promoter activity upon glucocorticoids treatment that was abolished by the glucocorticoid receptor antagonist RU-486. Single and double mutations of the two putative glucocorticoid response element sites within the promoter reduced the dexamethasone effect, suggesting that both glucocorticoid response elements are functionally active and required for induction. Our study shows that glucocorticoids exert a protective effect on stress-induced mesangial cell apoptosis in vitro and in vivo by upregulating NCDase and SphK1 expression and activity, resulting in enhanced levels of the protective lipid second messenger S1P.

Porcine circovirus type 2 DNA influences cytoskeleton rearrangements in plasmacytoid and monocyte-derived dendritic cells

Functional disruption of dendritic cells (DC) is an important strategy for viral pathogens to evade host defences. In this context, porcine circovirus type 2 (PCV2), a single-stranded DNA virus, impairs plasmacytoid DC (pDC) and conventional DC activation by certain viruses or Toll-like receptor (TLR) ligands. This inhibitory capacity is associated with the viral DNA, but the impairment does not affect all signalling cascades; TLR7 ligation by small chemical molecules will still induce interleukin-6 (IL-6) and tumour necrosis factor-α secretion, but not interferon-α or IL-12. In this study, the molecular mechanisms by which silencing occurs were investigated. PP2, a potent inhibitor of the Lyn and Hck kinases, produced a similar profile to the PCV2 DNA interference with cytokine secretion by pDC, efficiently inhibiting cell activation induced through TLR9, but not TLR7, ligation. Confocal microscopy and cytometry analysis strongly suggested that PCV2 DNA impairs actin polymerization and endocytosis in pDC and monocyte-derived DC, respectively. Altogether, this study delineates for the first time particular molecular mechanisms involved in PCV2 interference with DC danger recognition, which may be responsible for the virus-induced immunosuppression observed in infected pigs.

Preponderance of cells with stem cell characteristics in metastasising mouse mammary tumours induced by deregulated EphB4 and ephrin-B2 expression

We have previously shown that EphB4 and ephrin-B2 are differentially expressed in the mammary gland and that their deregulated expression in the mammary epithelium of transgenic mice leads to perturbations of the mammary parenchyma and vasculature. In addition, overexpression of EphB4 and expression of a truncated ephrin-B2 mutant, capable of receptor stimulation but incapable of reverse signalling, confers a metastasising phenotype on NeuT initiated mouse mammary tumours. We have taken advantage of this transgenic tumour model to compare stem cell characteristics between the non-metastasising and metastasising mammary tumours. We analysed the expression of the proliferation attenuating p21(waf) gene, which was significantly increased in the metastasising tumours. Moreover, we compared the expression of CK-19, Sca-1, CD24 and CD49f as markers for progenitor cells exhibiting a decreasing differentiation grade. Sca-1 expressing cells were the earliest progenitors detected in the non-metastasising NeuT induced tumours. The metastasising NeuT/EphB4 tumours were enriched in CD24 expressing cells, whereas the metastasising NeuT/truncated ephrin-B2 tumours contained in addition significant amounts of CD49f expressing cells. The same cell populations were also enriched in mammary glands of single transgenic MMTV-EphB4 and MMTV-truncated ephrin-B2 females indicating that deregulated EphB4-ephrin-B2 signalling interferes with the homeostasis of the stem/progenitor cell pool before tumour formation is initiated. Since the same cell populations are enriched in the normal tissue, primary mammary tumours and metastases we conclude that these progenitor cells were the origin of tumour formation and that this change in the tumour origin has led to the acquisition of the metastatic tumour phenotype.

Nuclear cysteine cathepsin variants in thyroid carcinoma cells

The cysteine peptidase cathepsin B is important in thyroid physiology by being involved in thyroid prohormone processing initiated in the follicular lumen and completed in endo-lysosomal compartments. However, cathepsin B has also been localized to the extrafollicular space and is therefore suggested to promote invasiveness and metastasis in thyroid carcinomas through, e.g., ECM degradation. In this study, immunofluorescence and biochemical data from subcellular fractionation revealed that cathepsin B, in its single- and two-chain forms, is localized to endo-lysosomes in the papillary thyroid carcinoma cell line KTC-1 and in the anaplastic thyroid carcinoma cell lines HTh7 and HTh74. This distribution is not affected by thyroid stimulating hormone (TSH) incubation of HTh74, the only cell line that expresses a functional TSH-receptor. Immunofluorescence data disclosed an additional nuclear localization of cathepsin B immunoreactivity. This was supported by biochemical data showing a proteolytically active variant slightly smaller than the cathepsin B proform in nuclear fractions. We also demonstrate that immunoreactions specific for cathepsin V, but not cathepsin L, are localized to the nucleus in HTh74 in peri-nucleolar patterns. As deduced from co-localization studies and in vitro degradation assays, we suggest that nuclear variants of cathepsins are involved in the development of thyroid malignancies through modification of DNA-associated proteins.

Intra-arterial injection of neural stem cells using a microneedle technique does not cause microembolic strokes

Intra-arterial (IA) injection represents an experimental avenue for minimally invasive delivery of stem cells to the injured brain. It has however been reported that IA injection of stem cells carries the risk of reduction in cerebral blood flow (CBF) and microstrokes. Here we evaluate the safety of IA neural progenitor cell (NPC) delivery to the brain. Cerebral blood flow of rats was monitored during IA injection of single cell suspensions of NPCs after stroke. Animals received 1 × 10(6) NPCs either injected via a microneedle (microneedle group) into the patent common carotid artery (CCA) or via a catheter into the proximally ligated CCA (catheter group). Controls included saline-only injections and cell injections into non-stroked sham animals. Cerebral blood flow in the microneedle group remained at baseline, whereas in the catheter group a persistent (15 minutes) decrease to 78% of baseline occurred (P<0.001). In non-stroked controls, NPCs injected via the catheter method resulted in higher levels of Iba-1-positive inflammatory cells (P=0.003), higher numbers of degenerating neurons as seen in Fluoro-Jade C staining (P<0.0001) and ischemic changes on diffusion weighted imaging. With an appropriate technique, reduction in CBF and microstrokes do not occur with IA transplantation of NPCs.

Impact of differential P450c17 phosphorylation by cAMP stimulation and by starvation conditions on enzyme activities and androgen production in NCI-H295R cells

CYP17A1 plays a pivotal role in the biosynthesis of androgens in the adrenals and the gonads. Although this enzyme catalyzes two different reactions on one single active site, its specific activities are regulated independently. Although the 17alpha-hydroxylase activity is rather constant and regulated by gene expression, the 17,20-lyase activity varies significantly with the amount of cofactors or by protein phosphorylation. cAMP increases CYP17A1 expression, P450c17 phosphorylation, and androgen production. However, the exact mechanism(s) and the specific regulators of CYP17A1 remain unknown. Therefore, we studied the regulation of adrenal androgen biosynthesis in human adrenal H295R cells focusing on CYP17A1. We analyzed androgen production and P450c17 activities in H295R cells grown under normal and serum-free conditions and/or after stimulation with 8-bromoadenosine-cAMP. H295R cells grown in starvation medium produced more androgens and had decreased HSD3B2 expression and activity but increased P450c17-17,20-lyase activity and serine phosphorylation. Although starvation increased serine phosphorylation of P450c17 specifically, cAMP stimulation enhanced threonine phosphorylation exclusively. Time-course experiments revealed that a short cAMP stimulation augmented threonine phosphorylation of P450c17 but did not increase 17,20-lyase activity. By contrast, long cAMP stimulation increased androgen production through increased P450c17 activities by enhancing CYP17A1 gene expression. We conclude that serum withdrawal shifts steroidogenesis of H295R cells towards androgen production, providing a suitable model for detailed studies of androgen regulation. In addition, our study shows that starvation and cAMP stimulation regulate P450c17 phosphorylation differentially and that an increase in P450c17 phosphorylation does not necessarily lead to enhanced enzyme activity and androgen production.

Antiproliferative effects of antiestrogens and inhibitors of growth factor receptor signaling on endometrial cancer cells

In patients with advanced estrogen-dependent type I endometrial cancer (EC), pharmacological treatment with progestins or antiestrogens is recommended, but primary and secondary resistance are common. The aim of our study was to investigate single-agent and dual-agent therapeutic strategies in estrogen receptor-positive human EC cells.

Tandem mass spectrometry identification and LC-MS quantification of intact cytokinin nucleotides in K-562 human leukemia cells

We describe here a new reversed-phase high-performance liquid chromatography with mass spectrometry detection method for quantifying intact cytokinin nucleotides in human K-562 leukemia cells. Tandem mass spectrometry was used to identify the intracellular metabolites (cytokinin monophosphorylated, diphosphorylated, and triphosphorylated nucleotides) in riboside-treated cells. For the protein precipitation and sample preparation, a trichloroacetic acid extraction method is used. Samples are then back-extracted with diethyl ether, lyophilized, reconstituted, and injected into the LC system. Analytes were quantified in negative selected ion monitoring mode using a single quadrupole mass spectrometer. The method was validated in terms of retention time stabilities, limits of detection, linearity, recovery, and analytical accuracy. The developed method was linear in the range of 1-1,000 pmol for all studied compounds. The limits of detection for the analytes vary from 0.2 to 0.6 pmol.

Vapor bubble generation around gold nano-particles and its application to damaging of cells

We investigated vapor bubbles generated upon irradiation of gold nanoparticles with nanosecond laser pulses. Bubble formation was studied both with optical and acoustic means on supported single gold nanoparticles and single nanoparticles in suspension. Formation thresholds determined at different wavelengths indicate a bubble formation efficiency increasing with the irradiation wavelength. Vapor bubble generation in Bac-1 cells containing accumulations of the same particles was also investigated at different wavelengths. Similarly, they showed an increasing cell damage efficiency for longer wavelengths. Vapor bubbles generated by single laser pulses were about half the cell size when inducing acute damage.

Inhibition of the Nef regulatory protein of HIV-1 by a single-domain antibody

The Nef protein of HIV-1 is important for AIDS pathogenesis, but it is not targeted by current antiviral strategies. Here, we describe a single-domain antibody (sdAb) that binds to HIV-1 Nef with a high affinity (K(d) = 2 × 10(-9)M) and inhibited critical biologic activities of Nef both in vitro and in vivo. First, it interfered with the CD4 down-regulation activity of a broad panel of nef alleles through inhibition of the Nef effects on CD4 internalization from the cell surface. Second, it was able to interfere with the association of Nef with the cellular p21-activated kinase 2 as well as with the resulting inhibitory effect of Nef on actin remodeling. Third, it counteracted the Nef-dependent enhancement of virion infectivity and inhibited the positive effect of Nef on virus replication in peripheral blood mononuclear cells. Fourth, anti-Nef sdAb rescued Nef-mediated thymic CD4(+) T-cell maturation defects and peripheral CD4(+) T-cell activation in the CD4C/HIV-1(Nef) transgenic mouse model. Because all these Nef functions have been implicated in Nef effects on pathogenesis, this anti-Nef sdAb may represent an efficient tool to elucidate the molecular functions of Nef in the virus life cycle and could now help to develop new strategies for the control of AIDS.

Multiple drug hypersensitivity: normal Treg cell function but enhanced in vivo activation of drug-specific T cells

Up to 10% of patients with severe immune-mediated drug hypersensitivity reactions have tendencies to develop multiple drug hypersensitivities (MDH). The reason why certain individuals develop MDH and the underlying pathomechanism are unclear. We investigated different T cell subpopulations in MDH patients and compared them with patients allergic to a single drug and with healthy controls (HC).

Single application of low-dose mycophenolate mofetil-OX7-immunoliposomes ameliorates experimental mesangial proliferative glomerulonephritis

IgA nephropathy, one of the most frequent forms of glomerulonephritis, characterized by mesangial hypercellularity and glomerular extracellular matrix (ECM) expansion, often leads to end-stage renal disease over a prolonged period. We investigated whether antiproliferative treatment in a single low dose specifically targeted to the glomerular mesangium by immunoliposomes (ILs) results in an amelioration of mesangial proliferative glomerulonephritis in rats (anti-Thy1.1 nephritis). Mycophenolate mofetil (MMF) containing ILs was generated that targets the Thy1.1 antigen (OX-7) in rat mesangial cells. Treatment benefit of a single intravenous dose of these ILs given 2 days after disease induction was investigated by stereology, immunohistochemistry, and functional analyses (creatinine, albuminuria) until day +9 and was compared among untreated and free MMF-treated rats using six male Wistar rats per group. MMF-loaded OX7-IL prevented creatinine increase and albuminuria. Stereological analyses of MMF OX7-IL-treated animals yielded 30% reduction of mesangial cells on day +9 and a 40% reduction of glomerular ECM volume on day +5, compared with all of the other nephritic animals. Furthermore, at days +5 and +9 we observed decreased ECM content and decreased glomerular volume (day +5) in the MMF-OX7-IL-treated group compared with the nephritic group treated with free MMF. In conclusion, MMF-OX7-IL-based directed drug delivery represents a novel approach for treating mesangial cell-mediated forms of glomerulonephritis.